Of all the products used to prepare microscopic materials for their definitive mounting on slides, the dyes are the more difficult ones to be substituted. Their selection has been made over decades by specialized chemists and microscopists who have chosen with exquisite care the products suitable for each task. There is a delicate chemical balance between the stain and the organelles or cytoplasmic products that they are intended to color.

 After more than 100 years of experience, any proposed substitute is destined to be third rate, as opposed to the professional dyes, and many of these are absolutely irreplaceable. For more information search the Stain File (below).

 Most amateurs understand this, and, when really in need, if they can, they take recourse to the most common classic colorants as Hematoxylin, Eosin, Carmine, Methylene blue, Methyl green, Fuchsine, and the like. The big problem is to acquire them in small quantities and at reasonable prices. The Europeans have at least two resources (listed in the appendix).

 Nevertheless, the young person, and sometimes the not so young amateur microscopist, may wish to highlight nuclei, cytoplasm, cilia, cirri, plastids, and membranes to study them easily but without setting out, of course, to make a professional result, and to prepare professional formulae.

 It is for them for whom I have written up these notes.

This is an impossible picture. It is an illustration mixing  picture elements, enhanced with a little design when needed. It was composed in PhotoPaint  to show the most abundant  inhabitants in my microscopic jungle of Cladophora.

 
MATERIALS and METHODS USED

 Three years ago before moving to Durango, I wanted to keep some samples from the very varied microfauna that lived on or between a Cladophora tuff that inhabited my freshwater aquarium at Cancún. And I took the opportunity to try a pair of dyes and a probable mounting medium whose possible utility intrigued me.

 Cladophora is a chlorophyte with a thin and profusely branched filamentous thallus that sometimes invades like a plague the aquariums of the amateurs. My aquarium was not an exhibition aquarium but a source of biological materials, so the alga grew on the understanding that it had the right to balance its own development with the many other inhabitants of the aquarium which included a numerous population of guppies. These and their abundant young passed their time exploring the tuffs of weed probably to feed on the microfauna that lived between and on it, and also on the new growth of alga.

 In order to obtain the required sample, I introduced underwater a small part of the algae into a plastic tube (discarded from a photographic 35 mm film) I cut the contents and I removed the tube. 

 I filtered the water, without applying any type of anesthesia, covering the mouth with a piece of silk screen with a mesh of 70 microns, and I filled it with hot AFA, a fixative with alcohol, formalin and acetic acid.

 Unfortunately the fixation (and dehydration) squashes the thalli and turns them into unattractive flattened ribbons but it preserves very well the microfauna.

COLORATION with ALLURA RED

I washed out the fixative with 30% alcohol, and finally with demineralized water, and after discarding this, always making use of the silk mesh, I filled the tube with a solution of Allura Red (a dye which in the U.S.A. has the FD&C Red #40 code, and which is known in the European Union under the E129 number). The Allura Red is a dye approved for foods and sold in the form of a 2.8% aqueous solution. The commercial solution is extremely concentrated and I used a dilution of 6 drops of solution in 30 milliliters of water. I leave the materials in contact for 3 hours. (Probably one hour could be enough.)

 
The following treatment included the washing of the dye with water (two changes) and a progressive dehydration with 30%, 50%, 75% alcohol, (half an hour in each one) and 96% (of this last two changes, each of one full hour).

 It is very probable that the dehydration protocol has been excessively long, but the behavior of the nail enamel that I thought to use as a mounting media was unknown to me and I preferred to err on the safe side.

MOUNTING IN NAIL POLISH ENAMEL

 
I placed a heavy drop of nail enamel on a coverslide. I passed a piece of alga to the center of  one slide, I extended its branches with a pair of needles, and removed with an absorbent paper and very quickly most of the surrounding alcohol and turning down the coverslide I lowered it taking care with the material.

I put the preparation safe from dust and under a 10 g weight (a flat headed screw of 6 mm diameter by 2.0 cm. long, with two nuts screwed on) for 10 hours. On the following day the slides were hard enough to allow safe use even with the immersion objective. As a precaution I made an additional seal with the same enamel.

The mounting was extremely successful, and supports my recommendation for the use of the nail enamel as a synthetic resinous medium at least at the amateur level.

The preparations are now more than 2 years old and as it will be seen from the enclosed images they perfectly maintain the morphology of the subjects, and also retain the color of dye used.

Most of the protozoa that I found in this first sample were heliozoans of the species Actinosphaerium eichornii. Finding them was lucky because its morphology is extremely interesting, as demonstrated by the attached photos. I include the live organism for a comparison of the information given by both methods. 

Actinosphaerium eichhornii  the larger cell, and Actinophrys sol, both alive, to compare their sizes. The insert at right shows one axopod of A. eichhornii, with the characteristic beads of  cytoplasmic flow over the rigid axis.	Actinosphaerium  eichhornii, stained with Allura Red, which displays its cellular structures, including the multiple nuclei.

Pictures of other organisms were not taken then, but I believe that the onion epithelium presented in my article on the onion skin, which is still in the same condition, and the ones of Actinosphaerium, are sufficient to think that it would behave more or less like the Fast Green, which I discuss next.

FAST GREEN FCF

 
There are three other water soluble dyes; sold in Mexico and the U.S.A. for dietetic aims (other manufacturers sell similar products in the United Kingdom and the European Union).

 I prepared a sample with the same method  using Green #3. This was a dye very well known and used in professional microscopy: the Fast Green FCF (see the Appendix note on the probable present composition of the green dye).

 I had been lucky because this time (a month after the first sample), the alga was luxuriant and the population very diverse.

 The following pictures are a small gallery of the organisms found. Of those the most interesting one is Euplotes (that also prompted a special article) which not only shows very good anatomical details, but even allows differentiation of the macro and micronucleus characteristic of the species. Pictures of this organism are gathered in a special table at the end of this section.

 The other species with enormous representation in the sample was Actinophrys sol.

 Of course in the case of both heliozoans, the axopodia were fixed in different states of retraction. But no professional method can do better than that. There is a clear differentiation between ecto and endoplasm, and the nuclei and digestive vacuoles are clearly visible in both species.

An x 4 obj. view of some branches of the thallus, showing epiphytes and “neighbors” mostly Actinophrys.	One live Actinophrys, displaying its normal relationship with the algae. This is its hunting position.

The fixed Actinophrys. Also seen in the pictures are many epiphytic cyanophyta.

With the 100x objective, the cell anatomy is shown, you also see the epiphytic cyanophyta, which will be discussed in another article.

Four steps in the development of a  cyst. Probably a reproductive one.

A Stentor population was also extremely abundant. But the rough treatment applied was not the best one to fix them in good conditions of conservation. Nevertheless it is easy to discover the striated pellicle, as well as the peristomal ring and the cytostome and gullet, although the opacity of the voluminous cytoplasm hides the details of the long beaded nucleus.

A cluster of contracted Stentors fixed to a Cladophora thallus.

Three stentors showing pellicle striation.	The ciliated crown of membranelles around the peristomal field and the spiral cytostome of another stentor individual.

A Carchesium (Ciliatea, Peritrichida,) was also abundant. With shame I share a very bad picture of the little arbuscle and one contracted individual showing the characteristic horse-shoe shaped nucleus.

Carchesium (all the zooids are contracting independently) stopped in its contraction by the hot fixative.	A Carchesium zooid showing the well stained horse-shoe shape nucleus

Of course they were Bdelloids distributed throughout the preparation, but we know very well that these rotifers hardly lend themselves to being fixed in extension. Nevertheless the following figure shows that the internal anatomy, including the germovitellarium, are well preserved  and differentiated, telling us that many Monogononta, that could respond to anesthesia, could probably be fruitfully colored.

Philodina alive, taken through a COL-D3 filter	Philodina semi-contracted x 40 Obj.

Numerous eggs in many stages of development were adhered to filaments of the alga.

 To add a different example of the uses I found for my green dye, these are two samples of pollen, colored in Fast Green FCF and mounted in PVA-G. Of course the onion skin also behaves very well with it.

Pollen grains of Ipomoea colored with Fast Green and mounted in PVA-G, Obj. x 100.	Pollen of Petunia grandiflora, idem.

NOTE: The new web page of McCormick, the manufacturer of the dyes that I tried, declares the green one as a mixture of Yellow #5 (tartrazine) and Blue #1 (Blue brilliant). Apparently the Food & Drug Administration of the EU decided finally to prohibit the dietetic use of the Fast Green. I have not tried new samples and do not know what their behavior is. The sample that I still have and use, was declared at the time to be Fast Green FCF.

Euplotes euristomum. Two pictures selected from 10 focus levels showing the good differentiation of the long ribbon nucleus, and the little round micronucleus (especially in the first picture) under the frontal border.

BLUE BRILLIANT  and TARTRAZINE

The other two dyes are the blue brilliant and the tartrazine.

 Both were proven in different and smaller samples, fixed with Gala 20, and mounted in Glycerin, with more or less promising results.

The following images are one picture of a gastrotrich and one of paramecium, showing their structures stained with the blue brilliant. It must be tried more extensively. It is a promising dye.

Chaetonotus. A gastrotrich, Gala 20, Blue brilliant, mounted in glycerin

Paramecium, from the same slide

 The tartrazine is much less useful because it stains in a diffuse manner, hardly differentiating the nucleus, or other cytoplasmic structures.

A ciliate, stained with Blue Brilliant, in another similar preparation	Paramecium, stained with tartrazine	A little ciliate (x 100 obj.) stained with malachite green. But it took more than 8 hours to reach that level of staining. I do not recommend it (at least with these techniques)

CONCLUSIONS

 Four dietetic dyes, economical and easily found in any supermarket, can render useful services to the amateur. Not all microscopists, especially the young ones, can reach, by their price or its availability, the professional dyes.

 All amateur microscopists are used to working with methylene blue, which is sold for aquarium use; eosin, which is sold in pharmacies like a disinfectant, (see JMC article for reference) and gentian violet, (see my April 2004 article) also obtainable in pharmacies like a therapeutic for pathogen yeast infections. Adding to them the very well known Lugol’s Iodine (or the Rhode's fixative, see my September 2003 article), and Chinese (or Indian) Ink, that can be used for example like a substitute for Nigrosine, in the so called "negative" coloration of ciliates (see Deflandre), we reached the number of nine available dyes for beginner microscopists, that can cover an ample range of techniques.

 There are other dietetic dyes and lakes that must be tried, and other sources of colorants (e.g. the textile dyes). I hope that this article stimulates the search for the useful ones, and that those who attain some good results, have the kindness to share these with other amateur microscopists.

REFERENCES

The McCormick page   http://www.mccormick.com/productdetail.cfm?id=6036

Jean Marie Cavanihac, www.microscopies.com 

G. Deflandre:   XXX Microscopie Practique. Lechevalier, 1947, 430 pgs.

The Stain File,  http://home.primus.com.au/royellis/stains.html

Histology Stains: http://www.laddresearch.com/General_Catalog/Chapter_2/LMStains/lmstains.html

W. Dioni, 'A cheap and precise slicer for teaching botany' Micscape April 2004

APPENDIX

 European sources for professional dyes

Brunel Microscopes

Marcel Lecomte:  http://users.skynet.be/Champignons_passion/